Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication of organ transplantation characterized by abnormal proliferation of lymphoid cells in the setting of immunosuppression after transplantation. Epstein-Barr virus (EBV) is responsible for abnormal lymphocyte proliferation in 50–80% of PTLDs, particularly in early-onset disease. Latent membrane protein 1 (LMP1) is the chief oncogenic protein of EBV and has been shown to be critical in the transformation of human B cells by the virus. LMP1 may activate several cellular signal transduction pathways including MAPK/ERK, p38/JNK, PI3K/AKT and NF-kB/AP-1. The ability to usurp and dysregulate signaling pathways may explain how LMP1 promotes cell transformation, survival, and proliferation. Our laboratory has shown that LMP1 isolated from EBV-associated B cell lymphoma lines of PTLD patients contains gain-of-function mutations at AA212 (G-S) and AA366 (S-T) that result in sustained ERK signaling, c-Fos activation, and AP-1 activity. In this study, we asked whether these mutations, or other genetic alterations, are present in primary EBV+ PTLD tumors themselves.
DNA was isolated from formalin-fixed paraffin-embedded tissue sections of EBV+ PTLD tumors (n=8). Nested PCR was used to amplify LMP1, and the PCR products generated were cloned and sequenced. The presence or absence of gain-of-function mutations at AA212 (G-S) and AA366 (S-T) were assessed. 7 out of the 8 EBV+ PTLD tumors demonstrated both gain-of-function mutations in LMP1. Furthermore, 6 out of the 8 tumors contained an extra repeat of 8 amino acids within the LMP1 signaling tail corresponding to a putative JAK3 binding motif. We have previously identified this repeat in 3 of 6 EBV+ B cell lymphoma lines from PTLD patients. LMP1 was also cloned from the whole blood of a pediatric small bowel PTLD patient. In addition to these 2 mutations being present, the 8 amino acid repeat was also present but in triplicate, suggesting this motif may be crucial to the oncogenic activity of LMP1. In order to assess host cell mutations in EBV+ PTLD tumors, a qBiomarker Mutation PCR array was performed for the PI3K/AKT/mTOR pathway, known to be important in human malignancies. While mutations in PI3K/AKT/mTOR were identified in EBV+ B cell lymphoma lines from PTLD patients, there were significantly more mutations in the primary EBV+ PTLD tumors, with 25 distinct mutations found within the PTEN, PI3K, and STK11 molecules. 3 distinct mutations were identified in more than 1 tumor, and 4 distinct mutations were shared between a cell line and tumor.
Our findings clearly demonstrate that key gain-of-functions mutations in LMP1 detected in blood and cell lines are also detected in the primary tumor, suggesting a role in tumorigenesis and great potential as biomarkers of EBV+ PTLD. Moreover, host cell mutations may also contribute to dysregulation of key signal transduction pathways in EBV+ PTLD.